1. Field of the Invention
The present invention relates to the field of molecular biological techniques useful for the construction of recombinant DNA molecules. More particularly, the present invention relates to methods that use bipartite oligonucleotide primer/adapters and exonuclease treatment for the rapid, efficient and directional joining of two or more DNA molecules into any vector at a single restriction site. The field of the invention also relates to PCR subcloning, deletion mutant construction, site-directed mutagenesis, and oligonucleotide site insertion.
2. Related Art
The use of the polymerase chain reaction (PCR) in nucleic acid research has provided a convenient way to amplify and construct recombinant DNA. PCR is an in vitro method of nucleic acid synthesis by which a particular segment of DNA can be specifically replicated. The method involves two oligonucleotide primers that flank the DNA fragment to be amplified and repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase. These primers hybridize to opposite strands of the target sequence to form a construct, the primers being oriented so that DNA synthesis by the polymerase proceeds across the region between the primers. Since the extension products themselves are also complementary to and capable of binding primers, successive cycles of amplification essentially double the amount of DNA synthesized in the previous cycle. The result is an exponential accumulation of the target fragment, approximately 2.sup.n, where n is the number of cycles of amplification performed.
In many applications, the orientation of the DNA fragment in the PCR construct is crucial for gene expression. The incorporation of different restriction enzyme sites into both ends of a PCR product is a common technique used to achieve this goal.sup.1. However, restriction enzyme cleavage at the ends of PCR products is often inefficient, and in some cases compromised by the presence of internal sites within the insert target DNA.
In most cases, subcloning of PCR products is required for further manipulation. Subcloning of these products generally involves the incorporation of restriction sites at the ends of PCR products.sup.1,2, or blunt-ended ligation of PCR products into the vector.sup.3. However, cloning of PCR products is often not straightforward.
Several technical problems exist with the use of such techniques. Restriction sites at the ends of PCR products are often inefficiently cleaved by restriction enzymes because they are too close to the end of the DNA fragment. The PCR products lack part of the binding region for the restriction enzyme to contact the DNA template. The introduction of extraneous nucleotides at the 3' end of the amplified DNA fragments by Taq DNA polymerase.sup.3,4 leads to relatively low overall efficiency of the blunt-end ligation reaction. The incorporation of extraneous nucleotides would alter a reading frame and constructs a copy that includes the undesired nucleotide. The efficiency of blunt ended ligation is poor for the following reasons: i) the K.sub.m for the activity of T4 ligase on blunt-ended DNA is nearly 100 times higher than its K.sub.m on DNA with cohesive ends, thus, ligation of blunt-ended DNA requires a high concentration of enzyme and a high concentration of DNA ends (greater than 1 .mu.M), therefore very large amounts of the fragment to be cloned are needed, ii) during blunt-end ligation, a fraction of the plasmid vector will recircularize and contribute to non-recombinant backgrounds, and iii) because of the high concentration of the fragments to be cloned, many recombinant plasmids will contain more than one insert of foreign DNA. Furthermore, blunt ended cloning is directionless.
The T/A cloning system (Invitrogen) has been used to overcome the extra nucleotide problem at the 3' end. However, using this technique, an extra dAMP is automatically inserted. This generates additional problems especially in expression studies, primarily because it will alter the reading frame. Another approach has been to use cohesive end cloning (provided by the incorporation of restriction sites at the 5' end of PCR primers). While this provides an alternative to blunt-end cloning, this and the T/A cloning system require several steps of DNA fragment purification, an overnight ligase-dependent ligation and colony selection to determine the correct orientation of the insert and are labor intensive, time consuming and/or of low efficiency. It will usually take at least one day to prepare DNA fragments and another day for the ligase reaction.
Strategies for ligase-free cloning of PCR products have also been proposed in an attempt to overcome some of these problems. For example, the recombinant circle PCR (RCPCR) technique generates circular DNA through heterologous annealing of sequence-overlapped ends on different PCR products.sup.5,6 .These circular DNA forms can be transformed directly into bacteria without a ligation procedure. However, this method requires either multiple sets of PCR primers or PCR reamplification of sequence-overlapped molecules to splice insert and vector DNA together.sup.7. Also in these applications, both insert and vector DNA must be amplified. Vector DNA amplification adds to the limitation on the size of the DNA fragment that can be amplified by Taq DNA polymerase.
An alternate strategy is to construct sequence-specific, single-stranded ends on both PCR products of insert and vector ends.sup.8-10, then splice them through a sequence homologous annealing process. In most applications, single-stranded ends are generated by the 3' to 5' exonuclease activity of T4 DNA polymerase.sup.8,9 with the overlapped sequence specifically designed and incorporated into PCR primers for both insert as well as vector DNA amplification. A specified, unique length of 3' recessed ends is then created in the presence of specific dNTP(s) and T4 DNA polymerase, and the circular form of DNA, assembled through sequence overlapped ends, is ready for transformation.
Aslanidis et al..sup.8 relates to a procedure for preparing recombinant molecules employing PCR products and a PCR-amplified plasmid vector. The procedure includes a step wherein the 3'-terminal sequence is removed by the action of the (3'-5') exonuclease activity of T4 DNA polymerase in the presence of a specific dNTP, providing fragments with 5'-extending single-stranded (SS) tails of defined sequence and length. Therefore, this method is restricted to those vectors which have modified, defined end sequences.
Stoker et al..sup.9 relates to the cloning of PCR products using the construction of cohesive termini on PCR products using a T4 DNA polymerase. Specifically, T4 DNA polymerase was used to remove 3' terminal sequence in the PCR products thus constructing the cohesive AccI and XmaI termini. In the described reaction, the 3' to 5' exonuclease activity of T4 DNA is to be limited to removal of only bases G and C in order to prepare the desired recessed termini.
Kuijper et al. relates to the preparation of "Prime" cloning vectors. T4 DNA polymerase is employed to produce single-stranded ends of vector and insert DNA in the presence of dTTP and dATP, respectively.
These methods.sup.8,9,20 used to create single-stranded ends by T4 DNA polymerase incorporate specific nucleotide(s) in the reaction to stop exonuclease activity at a nucleotide of specific sequences. Kaluz.sup.10 appears to fix PCR insert ends on two restriction sites which are compatible with vector sites and ligate them together for directional cloning. All of these methods relate to single fragment subcloning, and none of them could be used with chimeric genes or to construct deletion mutants as described in the present invention. This is because, among other reasons, they require either defined end sequences on both insert and vector DNA ends or different restriction sites on the ends of vector and insert DNA for directional cloning.
An alternative way to produce single-stranded ends employs uracil DNA glycosylase (UDG). This enzyme cleaves all dUMPs which are incorporated into the PCR primers.sup.11. Some of the aforedescribed methods, like RCPCR, require either multiple PCR primer sets, vector amplification, or vector end sequence modifications. Others require a double restriction enzyme cleavage of vector and insert DNA followed by a ligation process for directional cloning.sup.9,10.
Lohff and Cease relates to the use of a 3' to 5' exonuclease to prepare blunt-ended PCR products for non-directional cloning into blunt-cut vectors.
PCR SOEing.sup.14,15 is a relatively useful technique employed to construct gene mutants, fusion genes and chimeric genes. PCR SOEing has also been used for site-directed mutagenesis, as described by Higuchi..sup.14,21 However, this technique requires at least two sequential PCR amplifications. Since Taq DNA polymerase has a mutation rate of about 0.04%, each time that an amplification round is required, this incorporation of incorrect nucleotides is amplified. In general, PCR SOEing is limited to DNA fragments of about 1 kb or less.
Several protocols utilizing ligase-free ligation of PCR products have been described.sup.5-8,11. These protocols still have at least one or more of the following disadvantages: vector amplification, vector sequence modification, multiple primer sets and sequential PCR reamplification(s).
Kaluz et al..sup.10 has reported directional cloning of PCR products without restriction enzyme cleavage of insert DNA fragments. They used a strategy similar to that of Stoker.sup.9 to construct cohesive termini for subcloning. However, both protocols require a ligase-dependent ligation procedure. Other protocols with a ligase-independent procedure require vector amplification to incorporate a specific sequence for annealing.sup.7,8,11. The Stoker.sup.9, Kaluz.sup.10, RCPCR.sup.5-7 and ligation-independent cloning methods.sup.8,11 do not allow the construction of chimeric products of the PCR `SOEing` variety for the above-stated reasons.
A need continues to exist for a technique of constructing recombinant DNA molecules that addresses the disadvantages of present techniques, e.g., vector amplification, enzymatic manipulation, lack of directional cloning, insertion of restriction enzyme sites at the ends of PCR products where cleavage is inefficient, sequential PCR amplifications that multiply error and the incorporation of additional nucleotides which changes a reading frame and provides incorrect copies.